Genetically modified microorganism for producing 3-hydroxyhexanedioic acid and/or (e)-hex-2-enedioic acid and production method for said chemicals

ABSTRACT

A genetically modified microorganism that can produce 3-hydroxyadipic acid and/or α-hydromuconic acid with a high yield; and a method of producing 3-hydroxyadipic acid and/or α-hydromuconic acid using the genetically modified microorganism, are disclosed. The genetically modified microorganism has an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, and has an enhanced enzymatic activity to catalyze a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, wherein, in the genetically modified microorganism, a dicarboxylic acid excretion carrier function is deleted or decreased.

TECHNICAL FIELD

The present invention relates to a genetically modified microorganism that abundantly produces 3-hydroxyadipic acid and/or α-hydromuconic acid, and to a method of producing 3-hydroxyadipic acid and/or α-hydromuconic acid using the genetically modified microorganism.

BACKGROUND ART

3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid) and α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid) are C₆ dicarboxylic acids. These dicarboxylic acids can be used as raw materials for the production of polyesters by polymerization with polyhydric alcohols or as raw materials for the production of polyamides by polymerization with polyfunctional amines. Additionally, a compound lactamized by adding ammonia to the end of such a dicarboxylic acid can be used as a raw material for a polyamide.

Examples of the literature relating to the production of a C₆ dicarboxylic acid using a microorganism include Patent Document 1 that describes a method of producing 3-hydroxyadipic acid, α-hydromuconic acid, and/or adipic acid using a polypeptide having an excellent activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. The Document describes a biosynthesis pathway through which these substances undergo an enzymatic reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. In Patent Document 1, any gene to be modified is limited to an intracellular reaction in the above-mentioned biosynthesis pathway, and there is no mention of transporting 3-hydroxyadipic acid, α-hydromuconic acid, and/or adipic acid out of a cell.

Patent Document 2 describes a method of producing dicarboxylic acid using a bacterium modified to increase the expression level of dicarboxylic acid excretion carrier genes yjP, yjjB, yeeA, and ynfM, and mentions succinic acid as an example of a C₄ dicarboxylic acid and adipic acid as an example of a C₆ dicarboxylic acid.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: EP 3719121 A1 -   Patent Document 2: JP 2017-216881 A

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a genetically modified microorganism for producing 3-hydroxyadipic acid and/or α-hydromuconic acid with a high yield; and a method of producing a substance using the modified microorganism, wherein the genetically modified microorganism is based on a microorganism in which a 3-oxoadipyl-CoA reducing enzyme gene is introduced, or the expression of the gene is enhanced, so that the enzyme activity is enhanced, and furthermore, in which a dicarboxylic acid excretion carrier is modified.

Means for Solving the Problem

The inventors have intensively studied to achieve the above-described object, and consequently have come to complete the present invention through the discovery that, contrary to the prediction by conventional technologies, a genetically modified microorganism in which a dicarboxylic acid excretion carrier function is deleted or decreased has an excellent ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid.

That is, the present invention provides the following:

(1) A genetically modified microorganism having an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, and having an enhanced enzymatic activity to catalyze a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, wherein, in the genetically modified microorganism, a dicarboxylic acid excretion carrier function is deleted or decreased.

(2) The genetically modified microorganism according to (1), wherein the deletion or decrease of the dicarboxylic acid excretion carrier function is caused by the deletion or decrease of the function of YjjP or a homolog thereof and/or YjjB or a homolog thereof.

(3) The genetically modified microorganism according to (2), wherein a yjjP gene or a homolog gene thereof and/or a yjjB gene or a homolog gene thereof is/are destroyed or deleted.

(4) The genetically modified microorganism according to (1), wherein the deletion or decrease of the dicarboxylic acid excretion carrier function is caused by the deletion or decrease of the function of YeeA or a homolog thereof and/or YnfM or a homolog thereof.

(5) The genetically modified microorganism according to (4), wherein a yeeA gene or a homolog gene thereof and/or a ynfM gene or a homolog gene thereof is/are destroyed or deleted.

(6) The genetically modified microorganism according to any one of (1) to (5), wherein the microorganism belongs to the genus Escherichia or Serratia.

(7) The genetically modified microorganism according to any one of (1) to (6), wherein, in the genetically modified microorganism, a gene encoding an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is introduced.

(8) A method of producing 3-hydroxyadipic acid and/or α-hydromuconic acid, comprising culturing the genetically modified microorganism according to any one of (1) to (7).

Effects of the Invention

The genetically modified microorganism can produce 3-hydroxyadipic acid and/or α-hydromuconic acid with a higher yield than a microorganism of the parent strain in which the dicarboxylic acid excretion carrier gene is not modified.

MODE FOR CARRYING OUT THE INVENTION

Below in the specification, 3-hydroxyadipic acid may be abbreviated as 3HA, and α-hydromuconic acid may be abbreviated as HMA. Additionally, 3-oxoadipyl-CoA may be abbreviated as 3OA-CoA, 3-hydroxyadipyl-CoA may be abbreviated as 3HA-CoA, and 2,3-dehydroadipyl-CoA may be abbreviated as HMA-CoA. An enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA may be hereinafter referred to as “3-oxoadipyl-CoA reductase”. An operon formed by the yjjP and yjjB genes in the presence of a single promoter may be referred to as yjjPB. The proteins encoded by the yjjP and yjjB genes may be referred to as YjjP and YjjB respectively, and a complex of these may be referred to as YjjPB. The protein encoded by the yeeA gene may be referred to as YeeA, and the protein encoded by the ynfM gene may be referred to as YnfM.

The microorganism according to the present invention has a gene modified so that the dicarboxylic acid excretion carrier function is deleted or decreased. Making the dicarboxylic acid excretion carrier function be deleted or decreased means making the dicarboxylic acid excretion activity be deleted or decreased. The method of making the function be deleted or decreased is not limited, and can be performed by destroying or deleting the dicarboxylic acid excretion carrier gene, for example, through the following: using a gene mutation agent, ultraviolet radiation, or the like to perform a gene mutation treatment on the nucleotide sequence of the gene or a homolog gene thereof (hereinafter, these are collectively referred to as a “dicarboxylic acid excretion carrier gene”) or the nucleotide sequence of the promoter or terminator region of the gene; performing a site-specific mutagenesis method or the like to delete part or all of the nucleotide sequence; introducing a frameshift mutation into the nucleotide sequence; inserting a stop codon into the nucleotide sequence; or the like. It is also possible to destroy or delete the gene, using a genetic recombination technology to remove all or part of the nucleotide sequence of the dicarboxylic acid excretion gene or the nucleotide sequence of the promoter or terminator region of the gene or to replace the nucleotide sequence with another nucleotide sequence. A method preferable among these is preferably to delete part or all of the nucleotide sequence of the dicarboxylic acid excretion carrier gene.

The dicarboxylic acid excretion carrier gene the function of which is to be deleted or decreased in the present invention can be easily obtained, for example, through performing a BLAST (Basic Local Alignment Search Tool) search on a public database in NCBI (National Center for Biotechnology Information), KEGG (Kyoto Encyclopedia of Genes and Genomes), or the like using the amino acid sequence of a known dicarboxylic acid excretion carrier as a query sequence, and referring to the gene that encodes the applicable sequence. Alternatively, the gene can be obtained by PCR using, as a primer, an oligonucleotide generated on the basis of the nucleotide sequence of a known dicarboxylic acid excretion carrier gene, and using, as a template, the genomic DNA of another organism.

The amino acid sequence identity between a known dicarboxylic acid excretion carrier and the protein of a homolog of the carrier is specifically 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.

In this regard, in the present invention, the term “sequence identity” means a ratio (percentage) of the number of identical amino acid or nucleotide residues relative to the total number of amino acid or nucleotide residues over the overlapping portion of an amino acid sequence alignment (including an amino acid corresponding to the translation start site) or a nucleotide sequence alignment (including the start codon), which is obtained by aligning two amino acid or nucleotide sequences with or without introduction of gaps for an optimal match, and is calculated by the following formula (1). In the formula (1), the length of a shorter sequence being compared is not less than 100 amino acids or not less than 300 bases; in cases where the length of the shorter sequence is less than 100 amino acids or less than 300 bases, the sequence identity is not defined. The sequence identity can be easily investigated using, for example, a default parameter on BLAST. Additionally, the sequence identity can also be determined using a similar function implemented in a software program such as Genetyx.

Sequence identity (%)=the number of matches (without counting the number of gaps)/the length of a shorter sequence (excluding the terminal gaps)×100  (1)

In the present invention, the dicarboxylic acid excretion carrier the function of which is to be deleted or decreased is preferably YjjP or a homolog thereof, YjjB or a homolog thereof, YeeA or a homolog thereof, and/or YnfM or a homolog thereof.

YjjP and YjjB are proteins presumed to be putative succinate exporters, and are specifically, for example, YjP (NCBI Protein ID: NP_418784, SEQ ID NO: 2) from Escherichia coli str. K-12 substr. MG1655, YjjB (NCBI Protein ID: NP_418783, SEQ ID NO: 4), a YjjP homolog (SEQ ID NO: 6) from Serratia grimesii strain NBRC13537, and a YjjB homolog (SEQ ID NO: 8). Examples of genes that encode these proteins include yjjP (NCBI Gene ID: 948812, SEQ ID NO: 1) from Escherichia coli str. K-12 substr. MG1655, and yjjB (NCBI Gene ID: 948811, SEQ ID NO: 3), a yjjP homolog (SEQ ID NO: 5) from Serratia grimesii strain NBRC13537, and a yjjB homolog (SEQ ID NO: 7). In this regard, the sequence identity between the amino acid sequences represented by SEQ ID NOs: 2 and 6 is calculated using a function of Genetyx (% Identity Matrix) in accordance with the formula (1), and is found to be 71.1%. The sequence identity between the amino acid sequences represented by SEQ ID NOs: 4 and 8 is calculated and found to be 75.8%.

It is known that YjjP and YjjB that coexist have a dicarboxylic acid excretion activity (Biosci Biotechnol Biochem 2017 September; 81 (9): 1837-1844). In particular, JP 2017-216881 A mentions that modifying a bacterium so that the expression of yjjP and yjjB, which are dicarboxylic acid excretion carrier genes, is increased makes it possible to enhance the ability of the bacterium to produce dicarboxylic acid. The document mentions a C₄ succinic acid, a C₆ adipic acid, and the like as dicarboxylic acids to be produced. Furthermore, the document mentions that YjjP and YjjB from Escherichia coli and Enterobactr aerogenes belonging to Enterobacteriaceae actually have a dicarboxylic acid excretion activity. Accordingly, those skilled in the art consider that it is predictable that deleting or decreasing the dicarboxylic acid excretion activity in a microorganism having an ability to produce a C₆ dicarboxylic acid leads to decreasing the ability of the microorganism to produce a C₆ dicarboxylic acid. However, as demonstrated in EXAMPLES in the specification, deleting the functions of YjjP and YjjB enhances the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid that are C₆ dicarboxylic acids, and deleting or decreasing the dicarboxylic acid excretion activity in a microorganism having an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid makes it possible to enhance the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, contrary to the prediction of those skilled in the art.

YeeA is a protein presumed to be a putative transporter, and specific examples of the protein include YeeA (NCBI Protein ID: NP 416512, SEQ ID NO: 91) from Escherichia coli str. K-12 substr. MG1655. Examples of genes that encode these proteins include yeeA (NCBI Gene ID: 946545. SEQ ID NO: 90) from Escherichia coli str. K-12 substr. MG1655.

YnfM is a protein presumed to be a putative membrane transport protein, and specific examples of the protein include YnfM (NCBI Protein ID: NP_416113, SEQ ID NO: 93) from Escherichia coli str. K-12 substr. MG1655. Examples of genes that encode these proteins include ynfM (NCBI Gene ID: 946138, SEQ ID NO: 92) from Escherichia coli str. K-12 substr. MG1655.

It is known that YeeA and YnfM have a dicarboxylic acid excretion activity (Biosci Biotechnol Biochem 2017 September; 81 (9): 1837-1844). In particular, JP 2017-216881 A mentions that modifying a bacterium so that the expression of yeeA or ynfM, which are dicarboxylic acid excretion carrier genes, is increased makes it possible to enhance the ability of the bacterium to produce dicarboxylic acid. Additionally, the document mentions a C₄ succinic acid, a C₆ adipic acid, and the like as dicarboxylic acids to be produced. Furthermore, the document mentions that YeeA and YnfM from Pantoea ananatis and Enterobactr aerogenes belonging to family Enterobacteriaceae actually have a dicarboxylic acid excretion activity. Accordingly, those skilled in the art consider that it is predictable that deleting or decreasing the dicarboxylic acid excretion activity in a microorganism having an ability to produce a C₆ dicarboxylic acid leads to decreasing the ability of the microorganism to produce a C₆ dicarboxylic acid. However, as demonstrated in EXAMPLES in the specification, deleting the functions of YeeA and YnfM enhances the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid that are C₆ dicarboxylic acids, and thus deleting or decreasing the dicarboxylic acid excretion activity in a microorganism having an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid makes it possible to enhance the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, contrary to the prediction of those skilled in the art.

A homolog of YjjP, a homolog of YjjB, a homolog of YeeA, or a homolog of YnfM, as a subject the function of which is to be deleted or decreased in the present invention, preferably has a succinic acid excretion activity. The fact that the protein has a succinic acid excretion activity can be confirmed by, for example, verifying that the homolog gene introduced into Escherichia coli strain AFP184 (WO 2005/116227) having an ability to produce succinic acid enhances succinic acid productivity.

In the present invention, examples of methods of enhancing the activity of the enzyme (3-oxoadipyl-CoA reductase) that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA include: a method in which the genes encoding the enzymes are introduced into a host microorganism; a method in which the number of copies of each of the genes is increased; a method in which the promoter region upstream of the coding region and the ribosome-binding sequence are modified in each of the genes; and the like. These methods may be carried out individually or in combination. The method of introducing a gene is not limited to a particular method, and examples of the method that can be used include a method in which the gene incorporated in an expression vector capable of autonomous replication in a microorganism is introduced into a host microorganism, and a method in which the gene is integrated into the genome of a microorganism.

One or more genes may be introduced. Moreover, introduction of a gene and enhancement of the expression of the gene may be combined.

When a gene encoding an enzyme to be expressed in the present invention is integrated into an expression vector or the genome of a host microorganism, the nucleic acid to be integrated into the expression vector or the genome is preferably composed of a promoter, a ribosome-binding sequence, the coding region of the gene, and a transcription termination sequence, and may additionally contain a gene that controls the activity of the promoter.

The promoter used in the present invention is not limited to a particular promoter, provided that the promoter drives expression of the enzyme in the host microorganism; examples of the promoter include gap promoter, trp promoter, lac promoter, tac promoter, and T7 promoter.

In the present invention, the expression vector to be used to introduce a gene and enhance the expression of a gene is not limited to a particular vector, provided that the vector is capable of autonomous replication in the microorganism; examples of the vector include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above vectors.

In cases where a nucleic acid for genome integration is used in the present invention to introduce a gene or to enhance the expression of a gene, the nucleic acid for genome integration can be introduced by site-specific homologous recombination. The method for site-specific homologous recombination is not limited to a particular method, and examples of the method include a method in which a Red recombinase and FLP recombinase are used (Proc Natl Acad Sci USA. 2000 Jun. 6; 97 (12): 6640-6645.), and a method in which λ Red recombinase and the sacB gene are used (Biosci Biotechnol Biochem. 2007 December; 71 (12):2905-1.).

The method of introducing the expression vector or the nucleic acid for genome integration is not limited to a particular method, provided that the method is for introduction of a nucleic acid into a microorganism; examples of the method include the calcium ion method (Journal of Molecular Biology, 53, 159 (1970)), and electroporation (N M Calvin, P C Hanawalt. J. Bacteriol, 170 (1988), pp. 2796-2801).

The reaction scheme 1 below shows an exemplary reaction pathway required for the production of 3-hydroxyadipic acid and/or α-hydromuconic acid. In this scheme, the reaction A represents a reaction that generates 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA. The reaction B represents a reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. The reaction C represents a reaction that generates 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. The reaction D represents a reaction that generates 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA. The reaction E represents a reaction that generates α-hydromuconic acid from 2,3-dehydroadipyl-CoA.

It is known that in cases where a microorganism has an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, the microorganism has an enzyme that catalyzes at least the reaction A in the biosynthesis pathway represented by the reaction scheme 1, (WO 2019/107516, JP 2013-535203 A, and US 2011/0,124,911 A1).

A reaction that generates 3-hydroxyadipic acid and α-hydromuconic acid from 3-oxoadipylCoA preferably has the biosynthesis pathway represented by the reaction scheme 1. That is, in cases where a genetically modified microorganism according to the present invention has an ability to produce 3-hydroxyadipic acid, a host microorganism for the genetically modified microorganism preferably has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A), an ability to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA (the reaction B), and an ability to generate 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA (the reaction D). In cases where a genetically modified microorganism according to the present invention has an ability to produce α-hydromuconic acid, a host microorganism for the genetically modified microorganism preferably has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A), an ability to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA (the reaction B), an ability to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA (the reaction C), and an ability to generate α-hydromuconic acid from 2,3-dehydroadipyl-CoA (the reaction E).

A genetically modified microorganism that abundantly produces 3-hydroxyadipic acid and/or α-hydromuconic acid can be obtained using, as a host microorganism, a microorganism having the above-mentioned biosynthesis pathway, in which microorganism, the function(s) of YjjP or a homolog thereof, YjjB or a homolog thereof, YeeA or a homolog thereof, and/or YnfM or a homolog thereof is/are deleted or decreased, and additionally, an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is enhanced.

Examples of microorganisms originally having an ability to produce 3-hydroxyadipic acid include the following microorganisms.

The genus Escherichia such as Escherichia fergusonii or Escherichia coli. The genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila, or Serratia nematodiphila.

The genus Pseudomonas such as Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans, or Pseudomonas chlororaphis subsp. aureofaciens.

The genus Hafnia such as Hafnia alvei.

The genus Corynebacterium such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, or Corynebacterium glutamicum.

The genus Bacillus such as Bacillus badius, Bacillus magaterium, or Bacillus roseus.

The genus Streptomyces such as Streptomyces vinaceus, Streptomyces karnatakensis, or Streptomyces olivaceus.

The genus Cupriavidus such Cupriavidus metallidurans, Cupriavidus necator, or Cupriavidus oxalaticus.

The genus Acinetobacter such as Acinetobacter baylyi or Acinetobacter radioresistens.

The genus Alcaligenes such as Alcaligenes faecalis.

The genus Nocardioides such as Nocardioides albus.

The genus Brevibacterium such as Brevibacterium iodinum.

The genus Delftia such as Delftia acidovorans.

The genus Shimwellia such as Shimwellia blattae.

The genus Aerobacter such as Aerobacter cloacae.

The genus Rhizobium such as Rhizobium radiobacter. The genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila, or Serratia nematodiphila.

Among these microorganisms originally having an ability to produce 3-hydroxyadipic acid, microorganisms belonging to the genus Escherichia or Serratia are preferably used in the present invention.

Examples of microorganisms presumed to originally have an ability to produce α-hydromuconic acid include the following microorganisms:

The genus Escherichia such as Escherichia fergusonii or Escherichia coli. The genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila, or Serratia nematodiphila.

The genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, or Pseudomonas chlororaphis subsp. aureofaciens.

The genus Hafnia such as Hafnia alvei.

The genus Bacillus such as Bacillus badius.

The genus Cupriavidus such Cupriavidus metallidurans, Cupriavidus numazuensis, or Cupriavidus oxalaticus.

The genus Acinetobacter such as Acinetobacter baylyi or Acinetobacter radioresistens.

The genus Alcaligenes such as Alcaligenes faecalis.

The genus Delftia such as Delftia acidovorans.

The genus Shimwellia such as Shimwellia blattae.

Among these microorganisms originally having an ability to produce α-hydromuconic acid, microorganisms belonging to the genus Escherichia or Serratia are preferably used in the present invention.

In cases where a genetically modified microorganism according to the present invention originally does not have an ability to produce 3-hydroxyadipic acid, introducing, into a microorganism, a suitable combination of nucleic acids encoding enzymes that catalyze the reactions A, B, and D makes it possible to afford the ability to produce the acid. In cases where a genetically modified microorganism according to the present invention originally does not have an ability to produce α-hydromuconic acid, introducing, into the microorganism, a suitable combination of nucleic acids encoding enzymes that catalyze the reactions A, B, C, and E makes it possible to afford the ability to generate the acid.

In the present invention, a microorganism that can be used as a host to obtain a genetically modified microorganism is not limited to any particular microorganism provided that the microorganism allows genetic modification. The microorganism is preferably a microorganism belonging to the genus Escherichia, Serratia, Hafnia, Pseudomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, Alcaligenes, Brevibacterium, Delftia, Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia, or Candida, more preferably a microorganism belonging to the genus Escherichia, Serratia, Hafnia, or Pseudomonas, particularly preferably a microorganism belonging to the genus Escherichia or Serratia.

Specific examples of the enzymes that catalyze the reaction B to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA include the polypeptides described in the following (a) to (c):

(a) a polypeptide composed of an amino acid sequence represented by any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22;

(b) a polypeptide composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22, except that one or several amino acids are substituted, deleted, inserted, and/or added, and having an enzymatic activity that catalyzes the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; and

(c) a polypeptide composed of an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 and having an activity to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.

Furthermore, an enzyme classified as 3-hydroxyacyl-CoA dehydrogenase into EC1.1.1.35 and an enzyme classified as 3-hydroxybutyryl-CoA dehydrogenase into EC1.1.1.157 can also be used as an enzyme having a 3-oxoadipyl-CoA reductase activity. Specific examples of enzymes that catalyze a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA include PaaH (NCBI Protein ID: NP_745425.1) from Pseudomonas putida strain KT2440, PaaH (NCBI Protein ID: NP_415913.1) from Escherichia coli str. K-12 substr. MG1655, DcaH (NCBI Protein ID: CAG68533.1) from Acinetobacter baylyi strain ADP1, PaaH (NCBI Protein ID: WP_063197120) from Serratia plymuthica strain NBRC102599, and polypeptide (NCBI Protein ID: WP_033633399.1) from Serratia nematodiphila strain DSM21420. Among these, the polypeptides described in (a) to (c) above are preferable.

For the polypeptide used in the present invention and composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22, except that one or several amino acids are substituted, deleted, inserted, and/or added, and having 3-oxoadipyl-CoA reductase activity, the range represented by the phrase “one or several” is preferably 10 or less, more preferably 5 or less, particularly preferably 4 or less, and most preferably one or two. In the case of amino acid substitution, the activity of the original polypeptide is more likely to be maintained when an amino acid(s) is/are replaced by an amino acid(s) with similar properties (so-called conservative substitution). That is, the physiological activity of the original polypeptide is often maintained when the amino acid(s) is/are replaced by an amino acid(s) with similar properties. Thus, the amino acid(s) is/are preferably replaced by an amino acid(s) with similar properties. That is, the 20 amino acids constituting naturally occurring proteins can be divided into groups with similar properties, such as neutral amino acids with a less polar side chain (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp); it is often the case that substitution between amino acids in the same group does not change the properties of the original polypeptide.

For the polypeptide used in the present invention and having an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 and having 3-oxoadipyl-CoA reductase activity, the sequence identity is preferably not less than 80%, more preferably not less than 85%, further preferably not less than 90%, still further preferably not less than 95%, yet further preferably not less than 97%, and even further preferably not less than 99%.

All the polypeptides represented by SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 as described above in (a) contain a common sequence 1, composed of 24 amino acid residues and represented by SEQ ID NO: 23, within a region from the 15th to the 38th amino acid residues from the N terminus (hereinafter, an amino acid residue at the n-th position from the N terminus may conveniently be represented by n “a.a.”; for example, the region from the 15th to the 38th amino acid residues from the N terminus may be thus simply represented by “I5 to 38 a.a.”). In the common sequence 1, Xaa represents an arbitrary amino acid residue; the 13 a.a. is preferably a phenylalanine or leucine residue; the 15 a.a. is preferably a leucine or glutamine residue; the 16 a.a. is preferably a lysine or asparagine residue; the 17 a.a. is a glycine or serine residue, more preferably glycine residue; the 19 a.a. is preferably a proline or arginine residue, and the 21 a.a. is preferably a leucine, methionine, or valine residue. The common sequence 1 corresponds to the region of the NAD+-binding residue and the surrounding amino acid residues. In the NAD+-binding residues, the 24th amino acid residue in the common sequence 1 is aspartic acid, as described in Biochimie. 2012 February; 94 (2): 471-8., but in the common sequence 1, the residue is asparagine, which is characteristic. It is thought that the polypeptides represented by SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 exhibit excellent enzymatic activity as 3-oxoadipyl-CoA reductases due to the presence of the common sequence 1.

The polypeptides as described above in (b) and (c) also preferably contain the common sequence 1, composed of 24 amino acid residues and represented by SEQ ID NO: 23, within a region from 1 to 200 a.a. The common sequence is more preferably contained within a region from 1 to 150 a.a., further preferably from 1 to 100 a.a.

The nucleic acids encoding the polypeptides described in (a) to (c) according to the present invention may contain an additional sequence that encodes a peptide or protein added to the original polypeptides at the N terminus and/or the C terminus. Examples of such a peptide or protein can include secretory signal sequences, transport proteins, binding proteins, tag peptides applicable for purification, and fluorescent proteins. Among those peptides or proteins, a peptide or protein with a desired function can be selected depending on the purpose and added to the polypeptides of the present invention by those skilled in the art. It should be noted that the amino acid sequence of such a peptide or protein is not included in the calculation of sequence identity.

The nucleic acids encoding the polypeptides represented by SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 are not particularly limited, provided that those nucleic acids are composed of nucleotide sequences which can be translated into the amino acid sequences represented by SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22, and the nucleotide sequences can be determined by considering a set of codons (standard genetic code) corresponding to each amino acid. In this respect, the nucleotide sequences may be redesigned using codons that are frequently used by a host microorganism used in the present invention.

Specific examples of the nucleotide sequences of the nucleic acids that encode the polypeptides with the amino acid sequences represented by SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 include the nucleotide sequences represented by SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21, respectively.

In the present invention, whether or not a polypeptide encoded by a certain nucleic acid has 3-oxoadipyl-CoA reductase activity is determined as follows: transformant strains A and B described below are produced and grown in a culture test, and if the presence of 3-hydroxyadipic acid or α-hydromuconic acid in the resulting culture fluid is confirmed, it is judged that the nucleic acid encodes a polypeptide having 3-oxoadipyl-CoA reductase activity. The determination method will be described using the reaction scheme 1 above which shows a biosynthesis pathway.

The transformant strain A has enzymes that catalyze the reactions A, D, and E. The transformant strain B has enzymes that catalyze the reactions A, C, D, and E.

The transformant strain A is first produced. Plasmids for the expression of enzymes that catalyze the reactions A, D, and E, respectively, are produced. The reactions D and E can be catalyzed by an identical enzyme. The plasmids are introduced into Escherichia coli strain BL21 (DE3), which is a microorganism strain lacking abilities to produce both 3-hydroxyadipic acid and α-hydromuconic acid. An expression plasmid in which a nucleic acid encoding a polypeptide, which is a subject of analysis for the presence of the enzymatic activity of interest, is incorporated downstream of a suitable promoter is introduced to obtain the transformant strain A. The transformant strain A is cultured, and the post-culture fluid is examined for the presence of 3-hydroxyadipic acid. Once the presence of 3-hydroxyadipic acid in the culture fluid is confirmed, the transformant strain B is then produced. The transformant strain B is obtained by introducing a plasmid for the expression of an enzyme that catalyzes the reaction C into the transformant strain A. The transformant strain B is cultured, and the post-culture fluid is examined for the presence of α-hydromuconic acid. When the presence of α-hydromuconic acid in the post-culture fluid is confirmed, it indicates that 3-hydroxyadipic acid produced in the transformant strain A and α-hydromuconic acid produced in the transformant strain B are generated through production of 3-hydroxyadipyl-CoA, and so it is judged that the subject polypeptide has 3-oxoadipyl-CoA reductase activity.

Specific examples of a method that can be used as above-mentioned include a method described in WO 2019/107516.

The 3-oxoadipyl-CoA reductase activity value can be calculated by quantifying 3-hydroxyadipyl-CoA generated from 3-oxoadipyl-CoA used as a substrate by purified 3-oxoadipyl-CoA reductase, wherein the 3-oxoadipyl-CoA is prepared from 3-oxoadipic acid by an enzymatic reaction. The specific method is as follows.

3-Oxoadipic acid can be prepared by a known method (for example, a method described in Reference Example 1 of WO 2017/099209).

Preparation of 3-oxoadipyl-CoA solution: A PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase (pcaI and pcaJ; NCBI Gene IDs: 1046613 and 1046612) in the full-length form. The nucleotide sequences of primers used in this PCR are, for example, those represented by SEQ ID NOs: 24 and 25. The amplified fragment is inserted into the KpnI site of pRSF-1b (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution for 3-oxoadipyl-CoA preparation with the following composition, which is allowed to react at 25° C. for 3 minutes and then filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the enzyme, and the obtained filtrate is designated as 3-oxoadipyl-CoA solution.

(Enzymatic Reaction Solution)

100 mM Tris-HCl (pH 8.2)

10 mM MgCl₂

0.5 mM succinyl-CoA

5 mM 3-oxoadipic acid sodium salt

2 μM CoA transferase.

Identification of 3-oxoadipyl-CoA reductase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding 3-oxoadipyl-CoA reductase in the full-length form. The amplified fragment is inserted into the BamHI site of pACYCDuet-1 (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a 3-oxoadipyl-CoA reductase solution. The 3-oxoadipyl-CoA reductase activity can be determined by using the enzyme solution to prepare an enzymatic reaction solution with the following composition and quantifying 3-hydroxyadipyl-CoA generated at 25° C.

(Enzymatic Reaction Solution)

100 mM Tris-HCl (pH 8.2)

10 mM MgCl₂

150 μL/mL 3-oxoadipyl-CoA solution

0.5 mM NADH

1 mM dithiothreitol

10 μM 3-oxoadipyl-CoA reductase.

Next, specific examples of the enzymes that catalyze the reactions A and C to E are presented. As an enzyme that catalyzes the reaction A to generate 3-oxoadipyl-CoA, for example, an acyl transferase (s-ketothiolase) can be used. The acyl transferase is not limited by a particular number in the EC classification, and is preferably an acyl transferase classified into EC 2.3.1.-, specifically including an enzyme classified as 3-oxoadipyl-CoA thiolase and classified into EC 2.3.1.174, an enzyme classified as acetyl-CoA C-acetyltransferase and classified into EC 2.3.1.9, and an enzyme classified as acetyl-CoA C-acyl transferase and classified into EC 2.3.1.16. Among them, PaaJ from Escherichia coli str. K-12 substr. MG1655 (NCBI Protein ID: NP_415915), PcaF from Pseudomonas putida strain KT2440 (NCBI Protein ID: NP_743536), and the like can be suitably used.

Whether or not the above acyl transferases can generate 3-oxoadipyl-CoA from succinyl-CoA and acetyl-CoA used as substrates can be determined by measuring a decrease in NADH coupled with reduction of 3-oxoadipyl-CoA in a combination of the reaction to generate 3-oxoadipyl-CoA by purified acyl transferase and the reaction to reduce 3-oxoadipyl-CoA used as a substrate by purified 3-oxoadipyl-CoA reductase. The specific measurement method is, for example, as follows.

Identification of acyl transferase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding an acyl transferase in the full-length form. The amplified fragment is inserted into the SacI site of pACYCDuet-1 (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain an acyl transferase solution. The acyl transferase activity can be determined by using the enzyme solution to prepare an enzymatic reaction solution with the following composition and measuring a decrease in absorbance at 340 nm coupled with oxidation of NADH at 30° C.

100 mM Tris-HCl (pH 8.0)

10 mM MgCl₂

0.1 mM succinyl-CoA

0.2 mM acetyl-CoA

0.2 mM NADH

1 mM dithiothreitol

10 μg/mL 3-oxoadipyl-CoA reductase

5 μg/mL acyltransferase.

Whether an enzyme possessed by a microorganism has acyl transferase activity can be determined by performing the above-described measurement using CFE instead of purified acyl transferase. The specific measurement method targeted to E. coli is, for example, as follows.

Preparation of CFE: A loopful of E. coli strain MG1655 to be subjected to the measurement of the activity is inoculated into 5 mL of a culture medium (culture medium composition: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride) adjusted to pH 7, and incubated at 30° C. with shaking for 18 hours. The obtained culture fluid is added to 5 mL of a culture medium (culture medium composition: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride, 2.5 mM ferulic acid, 2.5 mM p-coumaric acid, 2.5 mM benzoic acid, 2.5 mM cis,cis-muconic acid, 2.5 mM protocatechuic acid, 2.5 mM catechol, 2.5 mM 3OA, 2.5 mM 3-hydroxyadipic acid, 2.5 mM α-hydromuconic acid, 2.5 mM adipic acid, 2.5 mM phenylethylamine) adjusted to pH 7, and incubated at 30° C. with shaking for 3 hours.

The obtained culture fluid is supplemented with 10 mL of 0.9% sodium chloride and then centrifuged to remove the supernatant from bacterial cells, and this operation is repeated three times in total to wash the bacterial cells. The washed bacterial cells are suspended in 1 mL of a Tris-HCl buffer consisting of 100 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol, and glass beads (with a diameter of 0.1 mm) are added to the resulting suspension to disrupt the bacterial cells at 4° C. with an ultrasonic disruptor. The resulting bacterial homogenate is centrifuged to obtain the supernatant, and 0.5 mL of the supernatant is filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the resulting filtrate, followed by application of 0.4 mL of the Tris-HCl buffer to the UF membrane, and this operation is repeated three times in total to remove low-molecular-weight impurities, and the resulting supernatant is then resuspended in the Tris-HCl buffer to a final volume of 0.1 mL, which is designated as CFE. Instead of purified enzyme, 0.05 mL of the CFE is added to a total of 0.1 mL of the enzymatic reaction solution to determine the enzymatic activity.

As an enzyme that catalyzes the reaction C to generate 2,3-dehydroadipyl-CoA, for example, an enoyl-CoA hydratase can be used. The enoyl-CoA hydratase is not limited by a particular number in the EC classification, and is preferably an enoyl-CoA hydratase classified into EC 4.2.1.-, specifically including an enzyme classified as enoyl-CoA hydratase or 2,3-dehydroadipyl-CoA hydratase and classified into EC 4.2.1.17. Among them, PaaF (NCBI Protein ID: NP_415911) from Escherichia coli str. K-12 substr. MG1655, PaaF (NCBI Protein ID: NP_745427) from Pseudomonas putida strain KT2440, and the like can be suitably used.

Since the reaction catalyzed by enoyl-CoA hydratase is generally reversible, whether or not an enoyl-CoA hydratase has an activity to catalyze a reaction that generates 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA used as a substrate can be determined by detecting 3-hydroxyadipyl-CoA generated using purified enoyl-CoA hydratase with 2,3-dehydroadipyl-CoA used as a substrate thereof, which is prepared from α-hydromuconic acid through an enzymatic reaction. The specific measurement method is, for example, as follows.

Preparation of α-hydromuconic acid: Preparation of α-hydromuconic acid can be performed according to the method described in Reference Example 1 of WO 2016/199858.

Preparation of 2,3-dehydroadipyl-CoA solution: A PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase (including pcaI and pcaJ; NCBI Gene IDs: 1046613 and 1046612) in the full-length form. The nucleotide sequences of primers used in this PCR are, for example, those represented by SEQ ID NOs: 24 and 25. The amplified fragment is inserted into the KpnI site of pRSF-1b (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution for 2,3-dehydroadipyl-CoA preparation with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the enzyme, and the obtained filtrate is designated as 2,3-dehydroadipyl-CoA solution.

Enzymatic Reaction Solution for 2,3-Dehydroadipyl-CoA Preparation

100 mM Tris-HCl (pH 8.0)

10 mM MgCl₂

0.4 mM succinyl-CoA

2 mM α-hydromuconic acid sodium salt

20 μg/mL CoA transferase.

Identification of enoyl-CoA hydratase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding an enoyl-CoA hydratase in the full-length form. The amplified fragment is inserted into the NdeI site of pET-16b (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-s-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain an enoyl-CoA hydratase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the enzyme. The enoyl-CoA hydratase activity can be confirmed by detecting 3-hydroxyadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MS/MS) (Agilent Technologies, Inc.).

100 mM Tris-HCl (pH 8.0)

10 mM MgCl₂

300 μL/mL 2,3-dehydroadipyl-CoA solution

1 mM dithiothreitol

20 μg/mL enoyl-CoA hydratase.

Whether or not an enzyme originally expressed in a host microorganism used in the present invention has enoyl-CoA hydratase activity can be determined by adding 0.05 ml, of the CFE, instead of purified enoyl-CoA hydratase, to a total of 0.1 mL of the enzymatic reaction solution and performing the above-described measurement. The specific CFE preparation method targeted to E. coli is as described for that used in determination of acyl transferase activity.

As an enzyme that catalyzes the reaction D to generate 3-hydroxyadipic acid and the reaction E to generate α-hydromuconic acid, for example, a CoA transferase or an acyl-CoA hydrolase, preferably a CoA transferase, can be used.

The CoA transferase is not limited by a particular number in the EC classification, and is preferably a CoA transferase classified into EC 2.8.3.-, specifically including an enzyme classified as CoA transferase or acyl-CoA transferase and classified into EC 2.8.3.6, and the like.

In the present invention, the term “CoA transferase” refers to an enzyme with activity (CoA transferase activity) to catalyze a reaction that generates carboxylic acid and succinyl-CoA from acyl-CoA and succinic acid used as substrates.

As an enzyme that catalyzes the reaction D to generate 3-hydroxyadipic acid and the reaction E to generate α-hydromuconic acid, PcaI and PcaJ from Pseudomonas putida strain KT2440 (NCBI Protein IDs: NP_746081 and NP_746082), and the like can be suitably used, among others.

Since the above enzymatic reactions are reversible, the CoA transferase activity against 3-hydroxyadipyl-CoA or 2,3-dehydroadipyl-CoA used as a substrate can be determined by detecting 3-hydroxyadipyl-CoA and 2,3-dehydroadipyl-CoA generated respectively using purified CoA transferase with 3-hydroxyadipic acid and succinyl-CoA, or α-hydromuconic acid and succinyl-CoA, used as substrates thereof. The specific measurement method is, for example, as follows.

Preparation of 3-hydroxyadipic acid: Preparation of 3-hydroxyadipic acid is performed according to the method described in Reference Example 1 of WO 2016/199856 A1.

Identification of CoA transferase activity using 3-hydroxyadipic acid as a substrate: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase in the full-length form. The amplified fragment is inserted into the KpnI site of pRSF-1 b (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-α-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the enzyme. The CoA transferase activity can be confirmed by detecting 3-hydroxyadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MS/MS) (Agilent Technologies, Inc.).

100 mM Tris-HCl (pH 8.0)

10 mM MgCl₂

0.4 mM succinyl-CoA

2 mM 3-hydroxyadipic acid sodium salt

20 μg/mL CoA transferase.

Preparation of α-hydromuconic acid: Preparation of α-hydromuconic acid can be performed according to a method described in Reference Example 1 of WO 2016/199858.

Identification of CoA transferase activity using α-hydromuconic acid as a substrate: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase in the full-length form. The amplified fragment is inserted into the KpnI site of pRSF-1b (manufactured by Merck Millipore) which is an expression vector for E. coli, in-frame with the histidine-tag sequence. The plasmid is introduced into E. coli BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5 mL 10K; manufactured by Merck Millipore) to remove the enzyme. The CoA transferase activity can be confirmed by detecting 2,3-dehydroadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MS/MS) (Agilent Technologies, Inc.).

100 mM Tris-HCl (pH 8.0)

10 mM MgCl₂

0.4 mM succinyl-CoA

2 mM α-hydromuconic acid sodium salt

20 μg/mL CoA transferase.

Whether or not an enzyme originally expressed in a host microorganism used in the present invention has CoA transferase activity can be determined by adding 0.05 mL of the CFE, instead of purified CoA transferase, to a total of 0.1 mL of the enzymatic reaction solution and performing the above-described measurement. The specific CFE preparation method targeted to E. coli is as described for that used in determination of acyl transferase activity.

When a nucleic acid encoding any one selected from the acyl transferase, the 3-oxoadipyl-CoA reductase, the enoyl-CoA hydratase, and the CoA transferase is introduced into a host microorganism in the present invention, the nucleic acid may be artificially synthesized based on the amino acid sequence information of the enzyme in a database, or isolated from the natural environment. In cases where the nucleic acid is artificially synthesized, the usage frequency of codons corresponding to each amino acid in the nucleic acid sequence may be changed depending on the host microorganism into which the nucleic acid is introduced.

In cases where the nucleic acids encoding the enzymes are isolated from the natural environment, the organisms as sources of the genes are not limited to particular organisms, and examples of the organisms include those of the genus Acinetobacter, such as Acinetobacter baylyi and Acinetobacter radioresistens; the genus Aerobacter, such as Aerobacter cloacae; the genus Alcaligenes, such as Alcaligenes faecalis; the genus Bacillus, such as Bacillus badius, Bacillus magaterium, and Bacillus roseus; the genus Brevibacterium, such as Brevibacterium iodinum; the genus Corynebacterium, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, and Corynebacterium glutamicum; the genus Cupriavidus, such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus numazuensis, and Cupriavidus oxalaticus; the genus Delftia, such as Delftia acidovorans; the genus Escherichia, such as Escherichia coli and Escherichia fergusonii; the genus Hafnia, such as Hafnia alvei; the genes Microbacterium, such as Microbacterium ammoniaphilum; the genus Nocardioides, such as Nocardioides albus; the genus Planomicrobium, such as Planomicrobium okeanokoites; the genus Pseudomonas, such as Pseudomonas azotoformans. Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas putida, and Pseudomonas reptilivora; the genus Rhizobium, such as Rhizobium radiobacter; the genus Rhodosporidium, such as Rhodosporidium toruloides; the genus Saccharomyces, such as Saccharomyces cerevisiae; the genus Serratia, such as Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia nematodiphila, Serratia odorifera, and Serratia plymuthica; the genus Shimwellia, such as Shimwellia blattae; the genus Streptomyces, such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus, and Streptomyces vinaceus; the genus Yarrowia, such as Yarrowia lipolytica; the genus Yersinia, such as Yersinia ruckeri; the genus Euglena, such as Euglena gracilis; and the genus Thermobifida, such as Thermobifida fusca; preferably those of the genera Acinetobacter, Corynebacterium, Escherichia, Pseudomonas, Serratia, Euglena, and Thermobifida.

In a microorganism according to the present invention, the function of pyruvate kinase is preferably deleted to enhance the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid.

Pyruvate kinase is classified in EC2.7.1.40, and is an enzyme that catalyzes a reaction by which phosphoenolpyruvate is dephosphorylated and converted into pyruvic acid and ATP. Specific examples include PykF (NCBI Protein ID: NP_416191, SEQ ID NO: 27) from Escherichia coli str. K-12 substr. MG1655, PykA (NCBI Protein ID: NP_416368, SEQ ID NO: 28), PykF (SEQ ID NO: 30), PykA (SEQ ID NO: 31) from Serratia grimesii strain NBRC13537, and the like. In cases where a microorganism used in the present invention has two or more genes encoding pyruvate kinase, all the pyruvate kinase functions are preferably deleted. Whether or not a polypeptide to be encoded by a gene possessed by a microorganism used in the present invention is pyruvate kinase can be checked by performing a BLAST search on the public database in NCBI, KEGG, or the like.

In a microorganism according to the present invention, the activity of phosphoenolpyruvate carboxykinase is preferably enhanced in order to enhance the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid.

Phosphoenolpyruvate carboxykinase is classified in EC4.1.1.49, and is an enzyme that catalyzes a reaction to generate oxaloacetic acid and ATP from phosphoenolpyruvate, carbon dioxide, and ADP. Specific examples include Pck (NCBI Protein ID: NP_417862, SEQ ID NO: 32) from Escherichia coli str. K-12 substr. MG1655, PckA_1 (SEQ ID NO: 33), PckA_2 (SEQ ID NO: 34) from Serratia grimesii strain NBRC13537, and the like.

From a physiological viewpoint, phosphoenolpyruvate carboxykinase serves for the main reaction in the production of glucose from fatty acid in gluconeogenesis. The reaction catalyzed by phosphoenolpyruvate carboxykinase is reversible. In the production of 3-hydroxyadipic acid and/or α-hydromuconic acid, the reaction progresses toward converting phosphoenolpyruvate and carbon dioxide into oxaloacetic acid.

Whether or not a polypeptide to be encoded by an enzyme gene used in the present invention is phosphoenolpyruvate carboxykinase can be checked by performing a BLAST search on a website of NCBI, KEGG, or the like. The genetically modified microorganism according to the present invention is cultured in a culture medium, preferably a liquid culture medium, containing, as a material for fermentation, a carbon source which can be used by ordinary microorganisms. The culture medium used contains, in addition to the carbon source that can be used by the genetically modified microorganism, appropriate amounts of a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins. Any of natural and synthetic culture media can be used as long as the medium contains the above-described nutrients.

The material for fermentation is a material that can be metabolized by the genetically modified microorganism. The term “metabolize” refers to conversion of a chemical compound, which a microorganism has taken up from the extracellular environment or intracellularly generated from a different chemical compound, to another chemical compound through an enzymatic reaction. Sugars can be suitably used as the carbon source. Specific examples of the sugars include monosaccharides, such as glucose, sucrose, fructose, galactose, mannose, xylose, and arabinose; disaccharides and polysaccharides formed by linking these monosaccharides; and saccharified starch solution, molasses, and saccharified solution from cellulose-containing biomass, each containing any of those saccharides.

The above-listed carbon sources may be used individually or in combination, and the culture is preferably performed in a culture medium containing glucose in particular. When a carbon source is added, the concentration of the carbon source in the culture medium is not particularly limited, and can be appropriately selected depending on the type of the carbon source. The concentration of the glucose is preferably 5 to 300 g/L.

As the nitrogen source used for culturing the genetically modified microorganism, for example, ammonia gas, aqueous ammonia, ammonium salts, urea, nitric acid salts, other supplementarily used organic nitrogen sources, such as oil cakes, soybean hydrolysate, cascin degradation products, other amino acids; vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, and bacterial cells and hydrolysate of various fermentative bacteria can be used. The concentration of the nitrogen source in the culture medium is not particularly limited, and is preferably from 0.1 g/L to 50 g/L.

As the inorganic salts used for culturing the genetically modified microorganism, for example, phosphoric acid salts, magnesium salts, calcium salts, iron salts, and manganese salts can be appropriately added to the culture medium and used.

The culture conditions for the genetically modified microorganism to produce 3-hydroxyadipic acid and/or α-hydromuconic acid are set by appropriately adjusting or selecting, for example, the culture medium with the above composition, culture temperature, stirring speed, pH, aeration rate, and inoculation amount, depending on, for example, the type of the genetically modified microorganism and external conditions.

The range of pH in a culture is not limited to any particular value provided that such a range makes it possible to grow the genetically modified microorganism, and is preferably a pH range of from 5 to 8, more preferably a pH range of from 5.5 to 6.8.

The range of the aeration condition in a culture is not limited to any particular value provided that such a range makes it possible to produce 3-hydroxyadipic acid and/or α-hydromuconic acid. To grow the microorganism mutant well, it is preferable that oxygen remain in the liquid phase or the gas phase in the culture container at least when the culture is started.

In cases where foam is formed in a liquid culture, an antifoaming agent such as a mineral oil, silicone oil, or surfactant may be appropriately added to the culture medium.

After a recoverable amount of 3-hydroxyadipic acid and/or α-hydromuconic acid is produced during culturing of the microorganism, the produced products can be recovered. The produced products can be recovered, for example isolated, according to a commonly used method, in which the culturing is stopped once a product of interest is accumulated to an appropriate level, and the fermentation product is collected from the culture. Specifically, the products can be isolated from the culture by column chromatography, ion exchange chromatography, activated charcoal treatment, crystallization, membrane separation, distillation or the like after separation of bacterial cells through centrifugation, filtration or the like. More specifically, examples include, but are not limited to, a method in which an acidic component is added to salts of the products, and the resulting precipitate is collected; a method in which water is removed from the culture by concentration using, for example, a reverse osmosis membrane or an evaporator to increase the concentrations of the products and the products and/or salts of the products are then crystallized and precipitated by cooling or adiabatic crystallization to recover the crystals of the products and/or salts of the products by, for example, centrifugation or filtration; and a method in which an alcohol is added to the culture to produce esters of the products and the resulting esters of the products are subsequently collected by distillation and then hydrolyzed to recover the products. These recovery methods can be appropriately selected and optimized depending on, for example, physical properties of the products.

EXAMPLES

The present invention will now be specifically described by way of Examples.

Reference Example 1

Production of a plasmid for expression of an enzyme that catalyzes a reaction to generate 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A) and an enzyme that catalyzes a reaction to generate 3HA-CoA from 3OA-CoA (the reaction B), a reaction to generate 3-hydroxyadipic acid from 3HA-CoA (the reaction D), and a reaction to generate α-hydromuconic acid from HMA-CoA (the reaction E)

The pBBR1MCS-2 vector, which is capable of autonomous replication in E. coli (ME Kovach, (1995), Gene 166: 175-176), was cleaved with XhoI to obtain pBBR1MCS-2/XhoI. To integrate a constitutive expression promoter into the vector, primers (SEQ ID NOs: 36 and 37) were designed for use in amplification of an upstream 200-b region (SEQ ID NO: 35) of gapA (NCBI Gene ID: NC_000913.3) by PCR using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pBBR1MCS-2/XhoI were ligated together using the “In-Fusion ID Cloning Kit” (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid extracted from the obtained recombinant E. coli strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::Pgap. Then, the pBBR1MCS-2::Pgap was cleaved with Seal to obtain pBBR1MCS-2::Pgap/ScaI. To amplify a gene encoding an enzyme that catalyzes the reaction A, primers (SEQ ID NOs: 39 and 40) were designed for use in amplification of the full length of the acyl transferase gene pcaF (NCBI Gene ID: 1041755; SEQ ID NO: 38) by PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pBBR1MCS-2::Pgap/ScaI were ligated together using the “In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::AT. Then, the pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT/HpaI. To amplify a gene encoding an enzyme that catalyzes the reactions D and E, primers (SEQ ID NOs: 43 and 44) were designed for use in amplification of a continuous sequence including the full lengths of genes together encoding a CoA transferase, pcaI and pcaJ (NCBI Gene IDs: 1046613 and 1046612, SEQ ID NOs: 41 and 42), by PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pBBR1MCS-2::AT/HpaI were ligated together using the In-Fusion HD Cloning Kit, and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::ATCT.

The pBBR1MCS-2::ATCT is cleaved with ScaI to obtain pBBR1MCS-2::ATCT/ScaI. To amplify a nucleic acid encoding a polypeptide represented by SEQ ID NO: 10, primers (SEQ ID NOs: 45 and 46) were designed for use in amplification of a nucleic acid represented by SEQ ID NO: 9 using the genomic DNA of Serratia marcescens strain ATCC13880 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pBBR1MCS-2::ATCT/ScaI were ligated together using the “In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::ATCTOR.

Reference Example 2

Production of a plasmid for expression of an enzyme that catalyzes a reaction to generate HMA-CoA from 3H A-CoA (the reaction C)

The pMW119 expression vector (manufactured by Nippon Gene Co., Ltd.), which is capable of autonomous replication in E. coli, was cleaved with SacI to obtain pMW119/SacI. To integrate a constitutive expression promoter into the vector, primers (SEQ ID NOs: 47 and 48) were designed for use in amplification of an upstream 200-b region (SEQ ID NO: 35) of gapA (NCBI Gene ID: NC_000913.3) by PCR using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pMW119/SacI were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant E. coli strain was confirmed in accordance with routine procedures, and the plasmid was designated as pMW119::Pgap. Then, the pMW119::Pgap was cleaved with SphI to obtain pMW119::Pgap/SphI. To amplify a gene encoding an enzyme that catalyzes the reaction C, primers (SEQ ID NOs: 50 and 51) were designed for use in amplification of the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 49) by PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pMW119::Pgap/SphI were ligated together using the “In-Fusion HD Cloning Kit” (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures. The obtained plasmid was designated as pMW119::EH.

Example 1

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is Deleted

To delete the YjjPB homolog function of a Serratia microorganism, a Serratia microorganism mutant in which the yjjPB homolog gene was deleted was produced.

A method of deleting a yjjPB homolog gene was performed in accordance with a method described in Proc Natl Acad Sci USA. 2000 Jun. 6; 97 (12): 6640-6645.

A PCR was performed using the following: a kanamycin-resistant gene to be used as a marker when a gene was deleted; a template that was pKD4 containing an FRT (FLP recognition target) sequence for the dropout of the kanamycin-resistant gene; and primers that were oligo DNAs of SEQ ID NOs: 52 and 53. Thus, PCR fragments having a sequence length of 1.6 kb for deletion of a yjjPB homolog gene were obtained. Into a Serratia grimesii strain NBRC13537, pKD46 that was a X-red recombinase expression plasmid was introduced to obtain an ampicillin-resistant strain. The resulting strain was inoculated into 5 mL of an LB culture medium containing 500 μg/mL ampicillin, and incubated at 30° C. with shaking for 1 day. A culture fluid in an amount of 0.5 mL was inoculated into 50 mL of an LB culture medium containing 500 μg/mL ampicillin and 50 mM arabinose, and cultured with rotation at 30° C. for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 5 μL of PCR fragments, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 30° C. with shaking for 2 hours. All of the resulting mixture was applied to an LB agar medium containing 25 μg/mL kanamycin, and incubated at 30° C. for 1 day. The resulting kanamycin-resistant strain was used for a colony direct PCR. The band length was used to verify the deletion of the gene of interest and the insertion of the kanamycin-resistant gene. The primers used were the oligo DNAs of SEQ ID NOs: 54 and 56.

Subsequently, the kanamycin-resistant strain was inoculated into 5 mL of an LB culture medium and subcultured at 37° C. twice to dropout pKD46, thereby obtaining an ampicillin-sensitive strain. Into the ampicillin-sensitive strain, pCP20 that was an FLP recombinase expression plasmid was introduced to obtain an ampicillin-resistant strain again. The resulting strain was subcultured at 40° C. twice, and then, subjected to a colony direct PCR. The band length was used to verify the dropout of the kanamycin-resistant gene. The primers used were the oligo DNAs of SEQ ID NOs: 55 and 56. The fact that the resulting strain was ampicillin-sensitive verified that pCP20 dropped out. The resulting strain was designated as SgΔyjjPB.

Example 2

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The plasmids produced in Reference Example 1 were each introduced into SgΔyjjPB produced in Example 1, whereby a Serratia microorganism mutant was produced.

The SgΔyjjPB was inoculated into 5 mL of an LB culture medium, and incubated at 30° C. with shaking for 1 day. The culture fluid in an amount of 0.5 mL was inoculated into 5 mL of an LB culture medium, and incubated at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 1 μL of pBBR1MCS-2::ATCTOR, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 30° C. with shaking for 1 hour. The resulting mixture in an amount of 50 μL was applied to an LB agar medium containing 25 μg/ml, kanamycin, and incubated at 30° C. for 1 day. The resulting strain was designated as SgΔyjjPB/3HA.

Reference Example 3

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The pBBR1 MCS-2::ATCTOR was introduced into a Serratia grimesii NBRC13537 in the same manner as in Example 2. The resulting strain was designated as Sg/3HA.

Example 3

Production Test of 3-Hydroxyadipic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Homolog Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The Serratia microorganism mutant produced in Example 2 was used to perform a test for 3-hydroxyadipic acid production.

A loopful of each mutant produced in Example 2 was inoculated into 5 mL of the culture medium 1 (10 g/L Bacto Tryptone (manufactured by Difco Laboratories), 5 g/L Bacto Yeast Extract (manufactured by Difco Laboratories), 5 g/L sodium chloride, 25 μg/mL kanamycin) adjusted to pH 7 (a glass test tube having a diameter of 18 mm with an aluminum plug), and incubated at 30° C. with shaking at 120 min⁻¹ for 24 hours. Subsequently, 0.25 mL of the culture fluid was added to 5 mL of the culture medium 11 (50 g/L glucose, 1 g/L ammonium sulfate, 50 mM potassium phosphate, 0.025 g/L magnesium sulfate, 0.0625 mg/L iron sulfate, 2.7 mg/L manganese sulfate, 0.33 mg/L calcium chloride, 1.25 g/L sodium chloride, 2.5 g/L Bacto Tryptone, 1.25 g/L Bacto Yeast Extract, 25 μg/mL kanamycin) adjusted to pH 6.5 (a glass test tube having a diameter of 18 mm with an aluminum plug), and stationarily incubated at 30° C.

Quantitative Analysis of a Substrate and a Product

The supernatant separated from bacterial cells by centrifugation of the culture fluid was processed by membrane treatment using Millex-GV (0.22 μm; PVDF; manufactured by Merck KGaA), and the resulting filtrate was analyzed according to the following method to measure the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. Furthermore, on the basis of the results, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the following formula (2), and are shown in Table 1.

Yield (%)=amount of product generated (mol)/amount of sugar consumed (mol)×100  (2)

Quantitative Analysis of 3-Hydroxyadipic Acid by LC-MS/MS

-   -   HPLC: 1290 Infinity (manufactured by Agilent Technologies, Inc.)

Column: Synergi hydro-RP (manufactured by Phenomenex Inc.), length: 100 mm, internal diameter: 3 mm, particle size: 2.5 μm

Mobile phase: 0.1% aqueous formic acid solution/methanol=70/30

Flow rate: 0.3 mL/min

Column temperature: 40° C.

LC detector: 1260DAD VL+ (210 nm)

-   -   MS/MS: Triple-Quad LC/MS (manufactured by Agilent Technologies,         Inc.)         Ionization method: ESI in negative mode

Quantitative Analysis of an Organic Acid by HPLC

-   -   HPLC: LC-10A (manufactured by Shimadzu Corporation)

Column: Shim-pack SPR-H (manufactured by Shimadzu GLC Ltd.), 250 mm in length, 7.8 mm in internal diameter, and 8 μm in particle size

Shim-pack SCR-101H (manufactured by Shimadzu GLC Ltd.), 250 mm in length, 7.8 mm in internal diameter, and 10 μm in particle size

Mobile phase: 5 mM p-toluenesulfonic acid

Reaction liquid: 5 mMp-toluenesulfonic acid, 0.1 mM EDTA, 20 mM Bis-Tris

Flow rate: 0.8 mL/min

Column temperature: 45° C.

Detector: CDD-10Avp (manufactured by Shimadzu Corporation)

Quantitative Analysis of Sugars and Alcohols by HPLC

-   -   HPLC: Shimadzu Prominence (manufactured by Shimadzu Corporation)

Column: Shodex Sugar SH1011 (manufactured by Showa Denko K.K.), length: 300 mm, internal diameter: 8 mm, particle size: 6 m

Mobile phase: 0.05M aqueous sulfuric acid solution

Flow rate: 0.6 mL/min

Column temperature: 65° C.

Detector: RID-10A (manufactured by Shimadzu Corporation)

Comparative Example 1

Production Test of 3-Hydroxyadipic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The mutant produced in Reference Example 3 was cultured in the same manner as in Example 3. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. Furthermore, on the basis of the results, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the formula (2), and are shown in Table 1.

Comparison between the results of Comparative Example 1 and those of Example 3 has revealed that the deletion of the YjjPB homolog function of a Serratia microorganism enhances the yield of 3-hydroxyadipic acid.

TABLE 1 Yield of Yield of Strain succinic acid (%) 3HA (%) Example 3 SgΔyjjPB/3HA 9.5 0.89 Comparative Sg/3HA 11 0.69 Example 1

Example 4

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is Deleted

To delete the YjjPB function of an Escherichia microorganism, an Escherichia microorganism mutant in which the yjjPB gene was deleted was produced.

A method of deleting a yjjPB gene was performed in accordance with a method described in Proc Natl Acad Sci USA. 2000 Jun. 6; 97 (12): 6640-6645.

A PCR was performed using the following: a template that was pKD4; and primers that were oligo DNAs of SEQ ID NOs: 57 and 58. Thus, PCR fragments having a sequence length of 1.6 kb for deletion of yjjPB were obtained. Into an Escherichia coli str. K-12 substr. MG1655, pKD46 that was an FRT recombinase expression plasmid was introduced to obtain an ampicillin-resistant strain. The resulting strain was inoculated into 5 mL of an LB culture medium containing 100 μg/mL ampicillin, and incubated at 37° C. with shaking for 1 day. A culture fluid in an amount of 0.5 mL was inoculated into 50 mL of an LB culture medium containing 100 μg/mL ampicillin and 50 mM arabinose, and cultured with rotation at 37° C. for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 5 μL of PCR fragments, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 37° C. with shaking for 2 hours. All of the resulting mixture was applied to an LB agar medium containing 25 μg/mL kanamycin, and incubated at 37° C. for 1 day. The resulting kanamycin-resistant strain was used for a colony direct PCR. The band length was used to verify the deletion of the gene of interest and the insertion of the kanamycin-resistant gene. The primers used were the oligo DNAs of SEQ ID NOs: 54 and 60.

Subsequently, the kanamycin-resistant strain was inoculated into 5 mL of an LB culture medium and subcultured at 40° C. twice to dropout pKD46, thereby obtaining an ampicillin-sensitive strain. Into the ampicillin-sensitive strain, pCP20 was introduced to obtain an ampicillin-resistant strain again. The resulting strain was subcultured at 43° C. twice, and then, subjected to a colony direct PCR. The band length was used to verify the dropout of the kanamycin-resistant gene. The primers used were the oligo DNAs of SEQ ID NOs: 59 and 60. The fact that the resulting strain was ampicillin-sensitive verified that pCP20 dropped out. The resulting strain was designated as EcΔyjjPB.

Example 5

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The plasmids produced in Reference Example 1 were each introduced into EcΔyjjPB produced in Example 4, whereby an Escherichia microorganism mutant was produced.

The EcΔyjjPB was inoculated into 5 mL of an LB culture medium, and incubated at 37° C. with shaking for 1 day. The culture fluid in an amount of 0.5 mL was inoculated into 5 mL of an LB culture medium, and incubated at 37° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 1 μL of pBBR1 MCS-2::ATCTOR and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 37° C. with shaking for 1 hour. The resulting mixture in an amount of 50 μL was applied to an LB agar medium containing 25 μg/mL kanamycin, and incubated at 37° C. for 1 day. The resulting strain was designated as EcΔyjjPB/3HA.

Reference Example 4

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The pBBR1MCS-2::ATCTOR was introduced into the Escherichia coli str. K-12 substr. MG1655 in the same manner as in Example 5. The resulting strain was designated as Ec/3HA.

Example 6

Production Test of 3-Hydroxyadipic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The mutant produced in Example 5 was cultured in the same manner as in Example 3. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the formula (2), and are shown in Table 2.

Comparative Example 2

Production Test of 3-Hydroxyadipic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The mutant produced in Reference Example 4 was cultured in the same manner as in Example 3. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the formula (2), and are shown in Table 2.

Comparison between the results of Comparative Example 2 and those of Example 6 has revealed that the deletion of the YjjPB function of an Escherichia microorganism enhances the yield of 3-hydroxyadipic acid.

TABLE 2 Yield of Yield of Strain succinic acid (%) 3HA (%) Example 6 EcΔyjjPB/3HA 8.8 1.2 Comparative Ec/3HA 14 1.1 Example 2

Example 7

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The plasmid pMW I19::EH produced in Reference Example 2 was introduced into the Serratia microorganism mutant produced in Example 2, whereby a Serratia microorganism mutant was produced.

The SgΔyjjPB/3HA was inoculated into 5 mL of an LB culture medium containing 25 μg/mL kanamycin, and incubated at 30° C. with shaking for 1 day. The culture fluid in an amount of 0.5 mL was inoculated into 5 mL of an LB culture medium containing 25 μg/mL kanamycin, and incubated at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 1 μL of pMW119::EH, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 30° C. with shaking for 1 hour. The resulting mixture in an amount of 50 μL was applied to an LB agar medium containing 500 μg/mL ampicillin and 25 μg/mL kanamycin, and incubated at 30° C. for 1 day. The resulting strain was designated as SgΔyjjPB/HMA.

Reference Example 5

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The pMW119::EH was introduced into the Sg/3HA in the same manner as in Example 7. The resulting strain was designated as Sg/HMA.

Example 8

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Homolog Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Example 7 was cultured in the same manner as in Example 3 except that a culture medium containing 25 μg/mL kanamycin and 500 μg/mil ampicillin was used. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. In this regard, α-hydromuconic acid was quantitated using an LC-MS/MS under the same conditions as 3-hydroxyadipic acid. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 3.

Comparative Example 3

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Homolog Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Reference Example 5 was cultured in the same manner as in Example 8. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 3.

Comparison between the results of Comparative Example 3 and those of Example 8 has revealed that the deletion of the YjjPB homolog function of a Serratia microorganism enhances the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

TABLE 3 Yield of Yield of Yield of Strain succinic acid (%) 3HA (%) HMA (%) Example 8 SgΔyjjPB/HMA 9.5 0.43 0.025 Comparative Sg/HMA 11 0.33 0.019 Example 3

Example 9

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The plasmid pMW119::EH produced in Reference Example 2 was introduced into the Escherichia microorganism mutant produced in Example 5, whereby an Escherichia mutant was produced.

The EcΔyjjPB/3HA was inoculated into 5 mL of an LB culture medium containing 25 μg/mL kanamycin, and incubated at 37° C. with shaking for 1 day. The culture fluid in an amount of 0.5 mL was inoculated into 5 mL of an LB culture medium containing 25 μg/ml, kanamycin, and incubated at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 1 μl, of pMW119::EH, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 37° C. with shaking for 1 hour. The resulting mixture in an amount of 50 μL was applied to an LB agar medium containing 100 μg/mL ampicillin and 25 μg/mL kanamycin, and incubated at 37° C. for 1 day. The resulting strain was designated as EcΔyjjPB/HMA.

Reference Example 6

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The pMW119::EH was introduced into the Ec/3HA in the same manner as in Example 9. The resulting strain was designated as Ec/HMA.

Example 10

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Example 9 was cultured in the same manner as in Example 6 except that a culture medium containing 25 μg/ml, kanamycin and 100 μg/mL ampicillin was used. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. In this regard, α-hydromuconic acid was quantitated using an LC-MS/MS under the same conditions as 3-hydroxyadipic acid. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 4.

Comparative Example 4

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Reference Example 6 was cultured in the same manner as in Example 10. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 4.

Comparison between the results of Comparative Example 4 and those of Example 10 has revealed that the deletion of the YjjPB function of an Escherichia microorganism enhances the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

TABLE 4 Yield of Yield of Yield of Strain succinic acid (%) 3HA (%) HMA (%) Example 10 EcΔyjjPB/HMA 9.9 1.6 0.053 Comparative Ec/HMA 19 1.1 0.032 Example 4

Example 11

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function and the Pyruvate Kinase Function are Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The pykF and pykA genes that encode pyruvate kinase in a Serratia microorganism were deleted to produce a Serratia microorganism mutant in which the pyruvate kinase function was deleted.

Production of a Serratia microorganism mutant in which pykF is deleted Genetic recombination was performed in the same manner as in Example 1 except that SgΔyjjPB was used as a subject from which pykF was to be deleted, that oligo DNAs of SEQ ID NOs: 61 and 62 were used as primers to obtain PCR fragments for deletion of pykF (SEQ ID NO: 29), that oligo DNAs of SEQ ID NO: 54 and 64 were used for colony direct PCR to verify the deletion of pykF and the insertion of a kanamycin-resistant gene, and that oligo DNAs of SEQ ID NOs: 63 and 64 were used for colony direct PCR to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as SgΔyjjPB,pykF.

Production of a Serratia Microorganism Mutant in which pykA is Deleted

Genetic recombination was performed in the same manner as in Example 1 except that SgΔjjPB,pykF was used as a subject from which pykA was to be deleted, that oligo DNAs of SEQ ID NOs: 66 and 67 were used as primers to obtain PCR fragments for deletion of pykA (SEQ ID NO: 65), that oligo DNAs of SEQ ID NO: 54 and 68 were used for colony direct PCR to verify the deletion of pykA and the insertion of a kanamycin-resistant gene, and that oligo DNAs of SEQ ID NOs: 68 and 69 were used for colony direct PCR to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as SgΔyjjPB,pykFA.

Production of a Serratia Microorganism Mutant in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The pBBR1MCS-2::ATCTOR generated in Reference Example 1 was introduced into the SgΔyjjPB,pykFA in the same manner as in Example 2. Next, the pMW119::EH was introduced into the resulting strain in the same manner as in Example 7. The resulting strain was designated as SgΔyjjPB,pykFA/HMA.

Reference Example 7

Production of a Serratia Microorganism Mutant in which the YjjPB Homolog Function is not Deleted, in which the Pyruvate Kinase Function is Deleted, and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

Genetic recombination was performed in the same manner as in Example 11 except that Serratia grimesii NBRC13537 was used as a subject from which pykF and pykA were to be deleted. The resulting strain was designated as SgΔpykFA/HMA.

Example 12

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Homolog Function and the Pyruvate Kinase Function are Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Example 11 was cultured in the same manner as in Example 8. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 5.

Comparative Example 5

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using a Serratia Microorganism Mutant in which the YjjPB Homolog Function is not Deleted, in which the Pyruvate Kinase Function is Deleted and, in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Reference Example 7 was cultured in the same manner as in Example 12. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 5.

Comparison between the results of Comparative Example 5 and those of Example 12 has revealed that the deletion of the YjjPB homolog function and pyruvate kinase function of a Serratia microorganism enhances the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

TABLE 5 Yield of Yield Yield succinic of 3HA of HMA Strain acid (%) (%) (%) Example 12 SgΔyjjPB, pykFA/HMA 41 2.0 0.11 Comparative SgΔpykFA/HMA 43 1.8 0.10 Example 5

Example 13

Production of an Escherichia Microorganism Mutant in which the YjjPB Function and the Pyruvate Kinase Function are Deleted

The pykF and pykA genes that encode pyruvate kinase in an Escherichia microorganism were deleted to produce an Escherichia microorganism mutant in which the pyruvate kinase function was deleted.

Production of an Escherichia microorganism mutant in which pykF is deleted Genetic recombination was performed in the same manner as in Example 4 except that EcΔyjjPB was used as a subject from which pykF was to be deleted, that oligo DNAs of SEQ ID NOs: 70 and 71 were used as primers to obtain PCR fragments for deletion of pykF (NCBI Gene ID: 946179, SEQ ID NO: 26), that oligo DNAs of SEQ ID NOs: 54 and 73 were used for colony direct PCR to verify the deletion of pykF and the insertion of a kanamycin-resistant gene, and that oligo DNAs of SEQ ID NOs: 72 and 73 were used for colony direct PCR to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as EcΔyjjPB,pykF.

Production of an Escherichia microorganism mutant in which pykA is deleted Genetic recombination was performed in the same manner as in Example 4 except that EcΔyjjPB,pykF was used as a subject from which PykA was to be deleted, that oligo DNAs SEQ ID NOs: 75 and 76 were used as primers to obtain PCR fragments for deletion of pykA (NCBI Gene ID: 946527, SEQ ID NO: 74), that oligo DNAs of SEQ ID NOs: 54 and 78 were used for colony direct PCR to verify the deletion of pykA and the insertion of a kanamycin-resistant gene, and that oligo DNAs of 77 and 78 were used for colony direct PCR to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as EcΔyjjPB,pykFA.

Reference Example 8

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted, in which the Pyruvate Kinase Function is Deleted, and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

Genetic recombination was performed in the same manner as in Example 13 except that Escherichia coli str. K-12 substr. MG1655 was used as a subject from which pykF and pykA were to be deleted. The resulting strain was designated as EcΔpykFA/HMA.

Example 14

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function and the Pyruvate Kinase Function are Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Example 13 was cultured in the same manner as in Example 10. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 6.

Comparative Example 6

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is not Deleted, in which the Pyruvate Kinase Function is Deleted, and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Reference Example 8 was cultured in the same manner as in Example 14. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 6.

Comparison between the results of Comparative Example 6 and those of Example 14 has revealed that the deletion of the YjjPB function and pyruvate kinase function of an Escherichia microorganism enhances the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

TABLE 6 Yield of Yield Yield succinic of 3HA of HMA Strain acid (%) (%) (%) Example 14 EcΔpykFA, yjjPB/HMA 25 4.8 0.17 Comparative EcΔpykFA/HMA 45 3.7 0.10 Example 6

Reference Example 9

Production of Plasmids to Enhance YjjPB Function

The pCDF-1b expression vector (manufactured by Merck Millipore), which is capable of autonomous replication in E. coli, was cleaved with HindIII and XbaI to obtain pCDF-1b/HindIII,XbaI. To integrate yjjP, yjjB, and promoter regions thereof, primers (SEQ ID NOs: 80 and 81) were designed for use in amplification of yjjP, yjjB, and promoter regions thereof (SEQ ID NO: 79) by PCR using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and a PCR reaction was performed in accordance with routine procedures. The resulting fragment and the pCDF-1b/HindIII,XbaI were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the resulting recombinant E. coli strain was confirmed in accordance with routine procedures, and the plasmid was designated as pCDF::yjjPB.

Reference Example 10

Production of an Escherichia Microorganism Mutant in which the YjjPB Function is Enhanced and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The plasmid pCDF::yjjPB produced in Reference Example 9 or pCDF-1b as a control was introduced into the Escherichia microorganism mutant produced in Reference Example 6, whereby an Escherichia mutant was produced.

The Ec/HMA was inoculated into 5 mL of an LB culture medium containing 25 μg/mL kanamycin and 100 μg/mL ampicillin, and incubated at 37° C. with shaking for 1 day. The culture fluid in amount of 0.5 mL was inoculated into 5 mL of an LB culture medium containing 25 μg/mL kanamycin and 100 μg/mL ampicillin, and incubated at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cell was washed with 10% (w/w) glycerol three times. The pellets washed were suspended in 100 μL of 10% (w/w) glycerol, and mixed with 1 μL of pCDF::yjjPB or pCDF-1b, and the resulting mixture was cooled on ice in an electroporation cuvette for 10 minutes. The resulting mixture was subjected to electroporation (at 3 kV, 200Ω, and 25 μF) using a “Gene pulser” (manufactured by Bio-Rad Laboratories, Inc.), and then immediately supplemented with 1 mL of an SOC culture medium, and the resulting mixture was incubated at 37° C. with shaking for 1 hour. The resulting mixture in an amount of 50 μL was applied to an LB agar medium containing 25 μg/mL kanamycin, 100 μg/mL ampicillin, and 50 μg/mL streptomycin, and incubated at 37° C. for 1 day. The resulting strains were designated as Ec/HMA,yjjPB and Ec/HMA,pCDF respectively.

Reference Example 11

Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by Using an Escherichia Microorganism Mutant in which the YjjPB Function is Enhanced and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, C, D, and E is Introduced

The mutant produced in Reference Example 10 was cultured in the same manner as in Example 10 except that a culture medium containing 25 μg/mL kanamycin, 100 μg/mL ampicillin, and 50 μg/mL streptomycin was used. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid. α-hydromuconic acid, and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid were calculated using the formula (2), and are shown in Table 7.

Comparison of the results of Reference Example 11 has revealed that the enhancement of the YjjPB function of an Escherichia microorganism increases the yield of succinic acid and decreases the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

TABLE 7 Yield of Yield of Yield of succinic 3HA HMA Strain acid (%) (%) (%) Reference Ec/HMA, yjjPB 13 1.6 0.026 Example 11 Ec/HMA, PCDF 16 0.38 0.0074

Example 15

Production of an Escherichia Microorganism Mutant in which the YeeA Function is Deleted

To delete the YeeA function of an Escherichia microorganism, an Escherichia microorganism mutant in which the yeeA gene was deleted was produced.

The yeeA gene was deleted in the same manner as in Example 4 except that oligo DNAs of SEQ ID NOs: 82 and 83 were used as primers to amplify a kanamycin-resistant gene and the FRT sequence using pKD4 as a template, that oligo DNAs of SEQ TD NOs: 54 and 85 were used as primers to verify the introduction of a kanamycin-resistant gene, and that oligo DNAs of SEQ ID NOs: 84 and 85 were used as primers to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as EcΔyeeA.

Example 16

Production of an Escherichia Microorganism Mutant in which the YeeA Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

In the same manner as in Example 5, the plasmid produced in Reference Example 1 was introduced into EcΔyeeA produced in Example 15, whereby an Escherichia microorganism mutant was produced. The resulting strain was designated as EcΔyeeA/3HA.

Example 17

Production Test of 3-Hydroxyadipic Acid by Using an Escherichia Microorganism Mutant in which the YeeA Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The mutant produced in Example 16 was cultured in the same manner as in Example 6. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the formula (2), and are shown in Table 8.

Comparison between the results of Comparative Example 2 and those of Example 17 has revealed that the deletion of the YeeA function of an Escherichia microorganism enhances the yield of 3-hydroxyadipic acid.

TABLE 8 Yield of Yield of Strain succinic acid (%) 3HA (%) Example 17 EcΔyeeA/3HA 5.8 1.6 Comparative Ec/3HA 14 1.1 Example 2

Example 18

Production of an Escherichia Microorganism Mutant in which the YnfM Function is Deleted

To delete the YnfM function of an Escherichia microorganism, an Escherichia microorganism mutant in which the YnfM gene was deleted was produced.

The ynfM gene was deleted in the same manner as in Example 4 except that oligo DNAs of SEQ ID NOs: 86 and 87 were used as primers to amplify a kanamycin-resistant gene and the FRT sequence using pKD4 as a template, that oligo DNAs of SEQ ID NOs: 54 and 89 were used as primers to verify the introduction of a kanamycin-resistant gene, and that oligo DNAs of SEQ ID NOs: 88 and 89 were used as primers to verify the dropout of the kanamycin-resistant gene. The resulting strain was designated as EcΔynfM.

Example 19

Production of an Escherichia Microorganism Mutant in which the YnfM Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

In the same manner as in Example 5, the plasmid produced in Reference Example 1 was introduced into EcΔynfM produced in Example 18, whereby an Escherichia microorganism mutant was produced. The resulting strain was designated as EcΔynfM/3HA.

Example 20

Production Test of 3-Hydroxyadipic Acid by Using an Escherichia Microorganism Mutant in which the YnfM Function is Deleted and in which a Plasmid that Expresses Enzymes that Catalyze the Reactions A, B, D, and E is Introduced

The mutant produced in Example 19 was cultured in the same manner as in Example 6. Measurements were made of the concentrations of the following: the 3-hydroxyadipic acid and other products accumulated in the culture supernatant; and the sugars remaining unused in the culture medium. On the basis of the values, the yields of 3-hydroxyadipic acid and succinic acid were calculated using the formula (2), and are shown in Table 9.

Comparison between the results of Comparative Example 2 and those of Example 20 has revealed that the deletion of the YnfM function of an Escherichia microorganism enhances the yield of 3-hydroxyadipic acid.

TABLE 9 Yield of Yield of Strain succinic acid (%) 3HA (%) Example 20 EcΔynfM/3HA 7.9 1.4 Comparative Ec/3HA 14 1.1 Example 2

Sequence Listing 

1. A genetically modified microorganism having an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, and having an enhanced enzymatic activity to catalyze a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, wherein, in the genetically modified microorganism, a dicarboxylic acid excretion carrier function is deleted or decreased.
 2. The genetically modified microorganism according to claim 1, wherein the deletion or decrease of the dicarboxylic acid excretion carrier function is caused by the deletion or decrease of the function of YjjP or a homolog thereof and/or YjjB or a homolog thereof.
 3. The genetically modified microorganism according to claim 2, wherein a yjjP gene or a homolog gene thereof and/or a yjjB gene or a homolog gene thereof is/are destroyed or deleted.
 4. The genetically modified microorganism according to claim 1, wherein the deletion or decrease of the dicarboxylic acid excretion carrier function is caused by the deletion or decrease of the function of YeeA or a homolog thereof and/or YnfM or a homolog thereof.
 5. The genetically modified microorganism according to claim 4, wherein a yeeA gene or a homolog gene thereof and/or a ynfM gene or a homolog gene thereof is/are destroyed or deleted.
 6. The genetically modified microorganism according to claim 1, wherein the microorganism belongs to the genus Escherichia or Serratia.
 7. The genetically modified microorganism according to claim 1, wherein, in the genetically modified microorganism, a gene encoding an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is introduced.
 8. A method of producing 3-hydroxyadipic acid and/or α-hydromuconic acid, comprising culturing the genetically modified microorganism according to claim
 1. 